This invention involves a procedure for preparation of the somatostatin analog, octreotide and its pharmaceutically acceptable salts formed by addition of acids or complexes of the same. Likewise, the invention is related to the preparation of intermediate compounds useful in the synthesis of octreotide in accordance with the invention.
While somatostatin possesses a very broad therapeutic potential and could be administered in a wide variety of clinical applications, its mean half-life in plasma is extremely short, reducing the number of applications possible. This drawback has promoted a number or research groups to establish the goal of developing more stable and more powerful analogs of somatostatin. One of these groups made a number of tests with cyclic octapeptides. One of these octapeptides yielded excellent biological activity both in vitro and in vivo (Pless J., Metabolism 41, 5-6 (1992)). This analog is Octreotide. Its structure is shown below: 
The presence of D-phenylalanine in the N-terminal end and an amino alcohol in the C-terminal end, along with the D-tryptophan residue and the disulfide bridge, make the molecule very resistant to metabolic degradation. The octreotide permits a 24 hour incubation in aggressive medium such as gastric juices or in intestinal mucosa.
Octreotide inhibits growth hormone for a lengthy period, inhibits the secretion of glucagon to a lesser degree, and inhibits insulin secretion only in a transient manner. It is thus more selective than other somatostatin analogues in regulating the levels of growth hormone in the body and therefore at present is indicated in acromegaly to control and reduce the plasma levels of such hormone. It is also used in the treatment of cellular alterations of gastroenteropancreatic endocrine origin and of certain types of tumors.
The primary ocreotide preparation described is a classic synthesis in solution (Bauer W., Pless J., (Sandoz) Eur. Pat. Appl. 29,579. Eidem U.S. Pat. No. 4,395,403 (1981, 1983). Syntheses in solid phase have been described subsequently (Mergler et al., Alsina et al., Neugebauer). In all of them, the objective is to form the entire peptide chain by solid phase peptide synthesis, starting the synthesis by the threoninol residue. This makes it mandatory to protect this residue.
The first author (Mergler M., Hellstern H., Wirth W., Langer W., Gysi P. and Prikoszovich W., Peltides: Chemistry and Biology; Proceedings of the 12th American Peptide Symposium. Smith, J. A. and Rivier J. E., Eds ESCOM, Leiden, Poster 292 Presentation (1991)) describes a synthetic process, using an aminomethyl resin upon which the Threoninol residue is incorporated with the two alcohol functions protected in acetal form. They carry out the synthesis following an Fmoc/tBu protection scheme, forming the disulfide bridge on resin by oxidation of the thiol groups of the previously deprotected cysteine residues and releasing and deprotecting the peptide with a 20% mixture of TFA/DCM.
In early 1997, Alsina J. et al. (Alsina J., Chiva C., Ortiz M., Rabanal F., Giralt E. and Albericio F., Tetrahedron Letters 38, 883-886 (1997)) described the incorporation, on active carbonate resins, of a Threoninol residue with the amino group protected by the Boc group and the side chain protected by a Bzl group. The synthesis was then continued by Boc/Bzl strategy. Formation of the disulfide bridge was carried out directly on resin using iodine, and the peptide was cleaved from the resin and its side chain protecting groups were simultaneously removed with HF/anisole 9/1. At a final stage the formyl group was removed with a piperidine/DMF solution. Neugebauer (Neugebauer W., Lefevre M. R., Laprise R., Escher E., Peptides: Chemistry, Structure and Biology, p. 1017, Marshal G. R. and Rivier J. E., Eds ESCOM, Leiden (1990)) described a linear synthesis with a yield of only 7%.
Edwards et al. (Edwards B. W., Fields C. G., Anderson C. J., Pajeau T. S., Welch M. J., Fields G. B., J. Med. Chem. 37 3749-3757 (1994)) carried out another solid-phase type approximation; they synthesized step-by-step on the resin, the peptide D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-HMP-resin. Next, they proceeded to form the disulfide on resin and then released the peptide from the resin by means of aminolysis with threoninol, with obtaining a total yield of only 14%.
All of these procedures carry out the formation of the disulfide bridge either on the totally deprotected peptide or on the resin.
This invention provides a procedure for obtaining octreotide and its pharmaceutically acceptable salts from acid addition, or complexes of same, by means of solid-phase synthesis upon polymer supports and with the intervention of protector groups of the Fmoc/tBu type, wherein it comprises the phases of:
1) Synthesis from the lineal peptide of seven amino acids
Boc-D-Phe-Cys-(Trt)-Phe-D-Trp-Lys(Boc)-Thr(tBu)-Cys(Trt)-Cl-trityl-R
said peptide being suitably protected (wherein the cysteines are protected with the trityl group, the lysine with a Boc group and the threonine with a tBu group) and anchored upon a 2-chloro trityl-R type resin wherein R is a polymer insoluble in DCM and DMF, reticulated polystyrene and the like.
2) Selective cleavage of the peptide-resin link without affecting neither the protecting groups of the terminal amino end nor the side chain protecting groups for the trifunctional amino acids, or
3a) Activation of the terminal carboxy group of the protected peptide and incorporation of the threoninol residue with no type of activation; and
4a) Formation of the disulfide bridge by oxidation with iodine; or
3b) Formation of the disulfide bridge by oxidation with iodine; and
4b) Activation of the terminal carboxy group of the protected peptide, with the disulfide bridge already formed, and incorporation of the threoninol residue with no type of protection.
5) Deprotection of the lateral chains and of the terminal amino end and obtaining octreotide.
6) Purification of the crude ocreotide by preparatory HPLC.
The second method according to this invention is summarized by the following diagram: 
The basic difference from the other procedures already described is that the introduction of the threoninol is carried out upon the protected peptidic structure (resin-free), which, when appropriately activated, leads quantitatively and without needing to make temporary protections or derivations upon the threoninol, to the protected precursor of octreotide, which in turn, with a simple acid treatment leads to octreotide with very high yields.
More specifically, this invention provides a procedure for obtaining octreotide based upon solid-phase synthesis with methodology of protecting groups of the type Fmoc/tBu upon a type-2-chloro trityl and the use of Boc-D-Phe for the terminal amino end of fragment 1-7 and subsequent incorporation of the threoninol residue as such.
The solid-phase synthesis is carried out using the 2-chloro trityl chloride resin (Barlos et al., Tetrahedron Letters 30, 3943-3946 (1989), Barlos et al., Tetrahedron Letters 30, 3947-3950 (1989)) incorporating in first place an Fmoc Cys (Trt). This support, due to its high steric hindrance, guarantees the incorporation of the Fmoc-Cys(Trt) residue without any racemization neither during the coupling itself nor during the subsequent basic treatments with 20% piperidine in DMF used to remove the Fmoc protecting group.
With the following peptidic skeleton (2-7) constructed:
Cys(Trt)-Phe-D-Trp-Lys-(Boc)-Thr(tBu)-Cys(Trt)-2-Cl-trityl-resin
this invention adds Boc-D-Phenylalanine to the terminal-N end of the peptidic chain, to obtain the linear skeleton:
xe2x80x83Boc-D-Phe-Cys(Trt)-D-Trp-Lys(Boc)-Thu(tBu)-Cys(Trt)-2-Cl-trityl-resin.
Subsequently, the peptide-resin is subjected to cleavage of the protected peptide fragment with acetic acide. The resulting product can either be cyclised by formation of a disulfide bridge with iodine in the same solution with simultaneous removal of the two trityl groups (3a), and subsequent incorporation of the threoninol residue (4a), or evaporated to a dry state to further proceed directly to the incorporation of the threoninol group by activation of the terminal carboxyl (3b) and subsequent oxidation of the entire peptide sequence.
The final step is always the removal of both the terminal amino protecting group of D-Phenylalanine (Boc) and the side chain protecting groups for Thr(t-Bu) and Lys(Boc) by means of a treatment with 70-95% TFA in presence of scavengers.
The crude octreotide is purified by HPLC and all of the homogeneous fractions are joined and lyophilized, thus obtaining octreotide in a 99% state of purity with a yield of the purification step of 60%.
Although it was at first expected that the activation of the C-terminal Cys residue required to couple the Threoninol would yield important amounts of (D-Cys)-octreotide, this reaction proceeded always with less than 1% epimerization. In short, this invention provides a procedure for obtaining octreotide, which is new and innovative in comparison with the synthetic strategies referring to already existing methods and described in the state of the art, with an overall synthesis and purification yield of greater than 40%.
The success of the invention lies in this feature which embodies a clear and competitive method for the synthesis of octreotide.
The abbreviations used in this description have the meanings set forth below: